Impact of Graft-Resident Leucocytes on Treg Mediated Skin Graft Survival.

The importance and exact role of graft-resident leucocytes (also referred to as passenger leucocytes) in transplantation is controversial as these cells have been reported to either initiate or retard graft rejection. T cell activation to allografts is mediated via recognition of intact or processed donor MHC molecules on antigen-presenting cells (APC) as well as through interaction with donor-derived extracellular vesicles. Reduction of graft-resident leucocytes before transplantation is a well-known approach for prolonging organ survival without interfering with the recipient's immune system. As previously shown by our group, injecting mice with IL-2/anti-IL-2 complexes (IL-2cplx) to augment expansion of CD4 T regulatory cells (Tregs) induces tolerance towards islet allografts, and also to skin allografts when IL-2cplx treatment is supplemented with rapamycin and a short-term treatment of anti-IL-6. In this study, we investigated the mechanisms by which graft-resident leucocytes impact graft survival by studying the combined effects of IL-2cplx-mediated Treg expansion and passenger leucocyte depletion. For the latter, effective depletion of APC and T cells within the graft was induced by prior total body irradiation (TBI) of the graft donor. Surprisingly, substantial depletion of donor-derived leucocytes by TBI did not prolong graft survival in naïve mice, although it did result in augmented recipient leucocyte graft infiltration, presumably through irradiation-induced nonspecific inflammation. Notably, treatment with the IL-2cplx protocol prevented early inflammation of irradiated grafts, which correlated with an influx of Tregs into the grafts. This finding suggested there might be a synergistic effect of Treg expansion and graft-resident leucocyte depletion. In support of this idea, significant prolongation of skin graft survival was achieved if we combined graft-resident leucocyte depletion with the IL-2cplx protocol; this finding correlated along with a progressive shift in the composition of T cells subsets in the grafts towards a more tolerogenic environment. Donor-specific humoral responses remained unchanged, indicating minor importance of graft-resident leucocytes in anti-donor antibody development. These results demonstrate the importance of donor-derived leucocytes as well as Tregs in allograft survival, which might give rise to new clinical approaches.

Distinct roles for major and minor antigen barriers in chimerism-based tolerance under irradiation-free conditions.

Eliminating cytoreductive conditioning from chimerism-based tolerance protocols would facilitate clinical translation. Here we investigated the impact of major histocompatibility complex (MHC) and minor histocompatibility antigen (MiHA) barriers on mechanisms of tolerance and rejection in this setting. Transient depletion of natural killer (NK) cells at the time of bone marrow (BM) transplantation (BMT) (20 × 106 BALB/c BM cells → C57BL/6 recipients under costimulation blockade [CB] and rapamycin) prevented BM rejection. Despite persistent levels of mixed chimerism, BMT recipients gradually rejected skin grafts from the same donor strain. Extending NK cell depletion did not improve skin graft survival. However, F1 (C57BL/6×BALB/c) donors, which do not elicit NK cell-mediated rejection, induced durable chimerism and tolerance. In contrast, if F1 donors with BALB/c background only were used (BALB/c×BALB.B), no tolerance was observed. In the absence of MiHA disparities (B10.D2 donors, MHC-mismatch only), temporal NK cell depletion established stable chimerism and tolerance. Conversely, MHC identical BM (BALB.B donors, MiHA mismatch only) readily engrafted without NK cell depletion but no skin graft tolerance ensued. Therefore, we conclude that under CB and rapamycin, MHC disparities provoke NK cell-mediated BM rejection in nonirradiated recipients whereas MiHA disparities do not prevent BM engraftment but impede skin graft tolerance in established mixed chimeras.

Treg-mediated prolonged survival of skin allografts without immunosuppression.

Injection of Interleukin-2 (IL-2) complexed with a particular anti-IL-2 monoclonal antibody (mab) JES6-1 has been shown to selectively expand CD4+Foxp3+ T regulatory T cells (Tregs) in vivo. Although the potency of this approach with regard to transplantation has already been proven in an islet transplantation model, skin graft survival could not be prolonged. Since the latter is relevant to human allograft survival, we sought to improve the efficiency of IL-2 complex (cplx) treatment for skin allograft survival in a stringent murine skin graft model. Here, we show that combining low doses of IL-2 cplxs with rapamycin and blockade of the inflammatory cytokine IL-6 leads to long-term (>75 d) survival of major histocompatibility complex-different skin allografts without the need for immunosuppression. Allograft survival was critically dependent on CD25+FoxP3+ Tregs and was not accompanied by impaired responsiveness toward donor alloantigens in vitro after IL-2 cplx treatment was stopped. Furthermore, second donor-type skin grafts were rejected and provoked rejection of the primary graft, suggesting that operational tolerance is not systemic but restricted to the graft. These findings plus the lack of donor-specific antibody formation imply that prolonged graft survival was largely a reflection of immunological ignorance. The results may represent a potentially clinically translatable strategy for the development of protocols for tolerance induction.

Oleic acid induces the novel apolipoprotein O and reduces mitochondrial membrane potential in chicken and human hepatoma cells.

Non-alcoholic fatty liver disease (NAFLD) is marked by hepatic fat accumulation and reflects a spectrum of chronic liver diseases associated with obesity, impaired insulin sensitivity and dyslipidemia. Apolipoprotein O (ApoO) is a new member of the plasma apolipoprotein family that may play a role in lipid metabolism and electron transport activity of the mitochondrium. However, its physiological functions have not been elucidated yet. Based on our previous data in a non-mammalian experimental system [1], we hypothesized that hepatic expression of ApoO is tightly linked not only to diet-induced hepatosteatosis, but also to increased lipoprotein-production induced by, e.g., hormones and oxidative stress. To gain insight into a mammalian experimental system, we compared the effects of lipid loading on ApoO regulation in chicken hepatoma LMH cells with those in the human hepatoma cell line HepG2. Incubation of the cells with BSA-complexed oleic acid (OA-Alb) induced triglyceride accumulation, but did not affect cell viability. qPCR using specific primer pairs and Western blot analysis with in-house produced rabbit anti-ApoO antisera demonstrated significant increase in ApoO transcript and protein levels in both cell lines. ROS formation due to OA-Alb treatment was only slightly altered in LMH cells, indicating an intact antioxidant defense system of the cells. Oxidative stress applied by addition of H2O2 revealed induction of ApoO transcript and protein level in the same or even higher extent as monitored in the presence of OA-Alb. Upon treatment with estrogen for 24 h quantitative analysis of ApoO transcript and protein revealed increases of ApoO expression supporting the assumption that estrogen affects lipoprotein metabolism at various points. Furthermore, both cell lines showed a significant decrease of the mitochondrial membrane potential upon incubation with OA-Alb. Therefore, we assume that our findings support a role of ApoO as an effector of compromised mitochondrial function that likely accompanies the onset of non-alcoholic fatty liver disease.

Hepatosteatosis and estrogen increase apolipoprotein O production in the chicken.

Apolipoprotein O (ApoO) is a recently discovered plasma apolipoprotein that may also play a role in the mitochondrial inner membrane. Possibly due to this complexity, its physiological functions have not been elucidated yet. To gain insight from a non-mammalian experimental system, we have investigated the regulation of ApoO levels in an alternative, well-suited model for studies on lipid metabolism, the chicken. qPCR using specific primer pairs and Western blot analysis with our rabbit anti-chicken ApoO antiserum demonstrated ApoO in the liver of chickens fed a control or a fat-enriched diet, as well as in 2 chicken hepatoma cell lines, LMH cells and the estrogen-responsive LMH-2A cells, under conditions of lipid loading by incubation with BSA-complexed oleic acid. Induced triglyceride accumulation in both the liver and the hepatic cells was associated with significantly increased levels of ApoO mRNA and protein. Furthermore, upon treatment for 24 h with estrogen of the estrogen receptor-expressing LMH-2A cells, quantitative analysis of ApoO transcripts and Western blotting revealed increases of ApoO expression. Finally, upon a single administration of estrogen to roosters that leads to hyperlipidemia, higher hepatic levels of both ApoO transcript and protein were observed within 24 h. Based on these data, we propose that hepatic expression of ApoO is tightly linked not only to diet-induced hepatosteatosis, but also to increased lipoprotein-production induced by, e.g., hormones. The findings support a role of ApoO as an effector of compromised mitochondrial function that likely accompanies the onset of non-alcoholic fatty liver disease.